Multipotent mesenchymal stem cells are recently discovered to be present in the adult bone marrow compartment. These mesenchymal stem cells are not only able to proliferate extensively in vitro, but also are able to differentiate into several mesenchymal cell lineages in vitro, including osteoblasts, chondrocytes, adipocytes, myoblasts and stromal fibroblasts. In vitro differentiation of these mesenchymal stem cells recapitulates complex differentiation processes of mesenchymal stem cells during embryonic and adult skeletal development. Thus discovery of these multipotent mesenchymal stem cells provides unprecedented opportunities to study cell fate determination at the molecular level. The ultimate goal of this research proposal is to understand the molecular mechanism of initiation of chondrogenic differentiation of mesenchymal stem cells. To achieve this, we would like to identify molecular switches (decision-making genes) that initiate chondrogenic differentiation of mesenchymal stem cells by a novel functional cloning strategy. Specifically, 1) we will construct a cDNA expression library using human mesenchymal cells at early stages of their differentiation; 2) we will transduce mouse mesenchymal stem cells with the cDNA expression library, screen for differentiated chondrocytes, clone, sequence and identify cDNA inserts from the differentiated chondrocytes; 3) we will validate regulatory function of the identified genes by re-expression of them individually in mesenchymal stem cells. Identification of key regulatory genes controlling chondrogenic differentiation of mesenchymal stem cells will help to elucidate the molecular mechanism underlying the complex process of chondrogenesis and will shed light on embryonic cell fate determination, skeletal pattern formation and cartilage development. Identification of such key regulatory genes may also provide opportunities to develop stem cell based therapies for treatment of chronic degenerative diseases such as osteoarthritis and acute cartilage damages such as facial, spine and joint injuries.